Directed Evolution Of Gene Encoding Alpha-amylase From Bacillus Licheniformis

Objective: to acquire higher activity strain of alpha-amylase . Methods: In this study,the gene encoding a hyperthermostable alpha-amylase from a bacillus licheniformis CICC10181 ,about 1580bp ,was cloned in pGEM-T vector containing lac promoter,and expressed in Escherichia coli cells. And finally we used error-prone PCR to randomly introduce mutations to the gene coding for the BLA. Because error-prone PCR is applicable to short fragment less than 1000bp, we cut alpha-amylase gene two parts by PCR,one 760bp, the other about 820bp. Subsequently, gene splicing by overlap extension (gene SOEing) was used to recombine alpha-amylase mutation gene. Finally, positive clones were screened on trypan blue culture medium and expressed in DH5α.At last, sequence analysis was carried out.Results: Six positive clone were acquired.Molecular weight of recombinant alpha-amylase was about 60 KD,which was fused by lacZ.Extracelluar production of the recombinant amylase was very little,which implied that putative signal peptide of this bacillus licheniformis alpha-amylase can not be strongly recognized by E.coli secretory system.This study recommended that the use ofthis signal peptides for extracelluar production of other foreign proteins in E.coli had better be cloned in expression vector,such as pET system vector.Error-prone PCR was used to mutate amylase gene randomly. Amylase activity was about 156U,a little higher than non-mutation amylase ,which was 101U. Molecular of weight recombinant amylase was also about 60 KD. And finally, sequencing amalysis shows that three points were mutated respectively,which were 163(C-G), 417(T-A),574(A-T).